Difference between revisions of "Part:BBa K3365061"
 
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<partinfo>BBa_K3365061 short</partinfo>
 
<partinfo>BBa_K3365061 short</partinfo>
  
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This part is a on-target detecting platform for dCas9-ω. We put this part in a plasmid and transfer it into E.coli, which was knocked out <i>rpoZ</i>. For convenience, we called it △<i>rpoZ</i>-MG1655. If dCas9-ω is binding to target sequence, the submit Omega fused to dCas9 C-terminal can activate the expression of reporter by recruiting the RNAP holoenzyme and the cell would emit red fluorescence,which means dCas9-ωis on-target, else the weak promoter and the lack of <i>rpoZ</i> makes the reporter cannot express or just have a quite low expression level.   
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This part is an on-target detecting platform for dCas9-ω. We put this part in a plasmid and transfer it into E.coli, which was knocked out <i>rpoZ</i>. For convenience, we called it △<i>rpoZ</i>-MG1655. If dCas9-ω is binding to target sequence, the subnit Omega fused to dCas9 C-terminal can activate the expression of reporter by recruiting the RNAP holoenzyme and the cell would emit red fluorescence,which means dCas9-ωis on-target, else the weak promoter and the lack of <i>rpoZ</i> makes the reporter cannot express or just have a quite low expression level.   
  
 
[[File:Target_binding_site_for_dCas9_and_subnit_Omega.png]]
 
[[File:Target_binding_site_for_dCas9_and_subnit_Omega.png]]
  
 
<b>Fig.1:</b> Schematic diagram of this part
 
<b>Fig.1:</b> Schematic diagram of this part
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We co-transformed plasmid contained this part and  pWJ66 with dCas9-ωin △<i>rpoZ</i>-MG1655 to determine if the system can work well. The transformed △<i>rpoZ</i>-MG1655 without pWJ66 is used as control.
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We took isolated colonies to pre-inoculate in 5mL of Luria-Bertani (LB) , incubating overnight in a shaker at 37℃. After that we detect the fluorescence intensity of the bacterial solution by microplate reader every two hours.Ths result is shown as follow.
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(please view:[https://parts.igem.org/File:The_result_of_microplate_reader.jpeg])
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[[File:The_result_of_microplate_reader.jpeg]]
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 13:11, 27 October 2020


Target transcription activating unit upstream of RFP

This part is an on-target detecting platform for dCas9-ω. We put this part in a plasmid and transfer it into E.coli, which was knocked out rpoZ. For convenience, we called it △rpoZ-MG1655. If dCas9-ω is binding to target sequence, the subnit Omega fused to dCas9 C-terminal can activate the expression of reporter by recruiting the RNAP holoenzyme and the cell would emit red fluorescence,which means dCas9-ωis on-target, else the weak promoter and the lack of rpoZ makes the reporter cannot express or just have a quite low expression level.

Target binding site for dCas9 and subnit Omega.png

Fig.1: Schematic diagram of this part

We co-transformed plasmid contained this part and pWJ66 with dCas9-ωin △rpoZ-MG1655 to determine if the system can work well. The transformed △rpoZ-MG1655 without pWJ66 is used as control. We took isolated colonies to pre-inoculate in 5mL of Luria-Bertani (LB) , incubating overnight in a shaker at 37℃. After that we detect the fluorescence intensity of the bacterial solution by microplate reader every two hours.Ths result is shown as follow. (please view:[1])

The result of microplate reader.jpeg

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 66
    Illegal NheI site found at 89
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 812
    Illegal XhoI site found at 803
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 667
    Illegal AgeI site found at 779
  • 1000
    COMPATIBLE WITH RFC[1000]